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Genetically engineered non-human mammal, construction method therefor and use thereof

Pending Publication Date: 2020-03-12
WANG MIAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a genetically engineered non-human mammal with a modified genome that includes the ApoE gene and various factors, such as SR-BI knockdown factors and vasoconstrictors. The non-human mammal can be derived from embryonic stem cells or fertilized eggs, and the exogenous polynucleotides are integrated into the genome using a tissue-specific promoter for the SR-BI knockdown factor and an inducible promoter for the vasoconstrictor. The invention also includes non-human mammalian cells, tissues, or organs derived from these animals. The invention further permits the use of these genetically engineered non-human mammals or their cells, tissues, or organs for screening or verifying drug efficacy.

Problems solved by technology

Therefore, the experimental data as collected according thereto are not accurate and thus cannot be used for research.
But beyond those, at present, there is no simple and effective AS model for the co-action of hyperlipidemia and hypertension.
This strategy is complicated, would trigger inflammation to mice from trauma, and is not convenient for administration regulation.
This strategy model is difficult to construct, and would produce relatively insignificant hyperlipidemia and atherosclerosis effects.
Mice are born with defects of hypertension and lipid metabolism, with hypertension being uncontrollable.

Method used

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Examples

Experimental program
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Effect test

example 1 preparation

of Exogenous Polynucleotide Sequences

[0065]Synthesizing a polynucleotide sequence to be inserted into the mice genome, comprising the following elements (named as an X polynucleotide sequence, as shown in SEQ ID NO. 8):

[0066]a polynucleotide sequence based on 5′ upstream end of exon 2 of the mice ApoE gene (with exon 2 locating in the antisense strand of chromosome 7 of mice: 19, 696, 109-19, 699, 188, having NCBI ID as 11816), the 5′ end being the start codon of exon 2; the element sequence is shown in SEQ ID NO. 2;

[0067]DM-PDZK1-flag, a polynucleotide sequence of the repressor protein of PDZK1 protein coding with flag tag, wherein the polynucleotide sequence, using a promoter of ApoE gene, reduces the level of SR-BI having activity in organisms at protein level; the element sequence is shown in SEQ ID NO. 3, wherein the sequence 5′-GATTACAAGGATGACGACGATAAG-3′ is the flag sequence;

[0068]Sh-m-SR-BI, a polynucleotide sequence with coding comprising siRNA which has a coding correspond...

example 2

Construction of Targeting Plasmid

[0075]The following polynucleotide elements were synthesized:

[0076]a polynucleotide sequence based on the 5′ end upstream of the mice ApoE gene to intron 1, with a FRT sequence, i.e. a FRT recombination site polynucleotide sequence, being inserted thereto; the element sequence is shown in SEQ ID NO. 1;

[0077]X polynucleotide sequence prepared in Example 1;

[0078]a downstream sequence of exon 4 based on mice ApoE gene; the element sequence is shown in SEQ ID NO. 7.

[0079]The above sequences are connected in tandem, and are connected into a plasmid vector carrying T7 promoter. The targeting plasmid as constructed is shown in FIG. 2.

example 3

Construction of Recombinant Mice

[0080]The construction steps are as follows:

[0081]1. Preparation of sgRNA

[0082]According to the design principle of sgRNA known in the art, for the ApoE gene sites, there are designed two polynucleotide sequences encoding sgRNA as follows:

(SEQ ID NO. 11)5′-GAAACCAGTCCGGGTTACTTGGG-3′(SEQ ID NO. 12)5′-GACCCAGCAAATACGCCTGCAGG-3′

[0083]wherein the sgRNA is directly obtained by artificial synthesis.

[0084]2. Preparation of Cas9 / RNA

[0085]Synthesizing Cas9 / RNA artificially: connecting the above sequences sgRNA into a recombinant plasmid vector carrying T7 promoter and the Cas9 expression sequence (Cas9 sequence is shown in SEQ ID NO. 16) for in vitro transcription, thereby obtaining microinjection Cas9 / RNA; and plasmid vector is Precut PCS plasmid available from Beijing Biocytogen Co., Ltd.

[0086]3. Preparation of Transgenic Mice

[0087]Microinjecting the prepared sgRNA and the Cas9 recombinant plasmid or the Cas9 / RNA recombinant plasmid, together with the target...

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Abstract

The present invention relates to genetically engineered non-human mammal and cells, organs and tissues; to use thereof in medicinal and disease research; to a method for producing non-human animals and cells, organs and tissues; and to a method for researching in medicine and disease by virtue of the non-human mammals or cells, organs or tissues.

Description

[0001]The present application claims the priority of the Chinese Patent Application No. 201710292460.X, entitled “Genetically Engineered Non-Human Mammal, and the Construction Method therefor and Use thereof”, filed on Apr. 28, 2017, which is incorporated herein by reference in its entirety.TECHNICAL FIELD[0002]The present invention relates to genetically engineered non-human mammal and cells, organs and tissues; to use thereof in medicinal and disease research; to a method for producing non-human animals and cells, organs and tissues; and to a method for researching in medicine and disease by virtue of the non-human mammals or cells, organs or tissues.BACKGROUNDS[0003]Atherosclerosis (AS) and cardiovascular death resulted therefrom, such as myocardial infarction and stroke, are the first reasons causing human death of disease. AS is a vascular disease caused by multiple factors, with lipid metabolism disorders, i.e. hyperlipidemia and hypertension, being major pathogenic risk-facto...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/90
CPCC12N2800/107C12N2517/02C12N2310/20C12N15/90C12N2800/60A01K67/0276A01K2217/075A01K2227/105A01K2267/0375A01K2217/077
Inventor WANG, MIAOCHEN, HONG
Owner WANG MIAO
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